Report for 9/3/2014 through 9/7/2015: Oversight and Support of Client-Specific Projects Over 2014-15 Feldman engaged in research projects with seven labs: four from NICHD, one from NCI, one from NHLBI and one from the Childrens National Medical Center. Zebrafish model of the human pediatric disease Smith-Lemli-Opitz Syndrome (SLOS). SLOS is an autosomal recessive, multiple malformation syndrome with pediatric onset characterized by intellectual disability and aberrant behavior. Several zebrafish lines are being investigated that carry mutant alleles of dhcr7, the zebrafish ortholog to human gene DHCR7. These lines were established through the NICHD Zebrafish Core via TALEN genome engineering during 2012-13. In 2013-14 and continuing in 2014-15, efforts have focused on defining phenotypes associated with these mutant alleles affecting metabolism, morphology, viability, fertility and behavior. In 2014-15, experiments were also conducted in which phenotypes were modulated by environmental and metabolic interventions. This project will continue in 2015-16. Function of zebrafish orthologs to human genes implicated in childhood gigantism. Gigantism arises due to excess growth hormone (GH) secretion during childhood, before the growth plates close. The zebrafish ortholog to a human gene implicated as a driver of gigantism was cloned and its effects (when transiently overexpressed) on zebrafish morphology and growth were investigated in 2012-13. In 2013-14 we began and in 2014-15 completed a description of this genes native expression in zebrafish. To understand whether chronic misexpression of this gene can drive hypertophic growth in zebrafish, we built a construct that expresses the gene in response to Gal4 and established zebrafish lines carrying this construct. This project will continue into 2015-16, during which period we willl cross these lines with lines that respectively express Gal4 in the pituitary, hypothalamus and throughout the CNS. Beginning in 2013-14 and continuing in 2014-15, we have worked to develop novel software and zebrafish holding systems to facilitate longitudinal growth measurements. This project will continue into 2015-16. Function of zebrafish orthologs to human genes implicated in familial adrenal hyperplasia. The following novel approach was initiated in 2012-13. To understand the molecular function of a particular human gene, ARMC5, implicated as a tumor suppressor for adrenal hyperplasia, global RNA sequencing was performed on zebrafish embryos in which the zebrafish ortholog to this gene, armc5, was (1) downregulated by antisense morpholino oligonucleotide injection, (2) upregulated by RNA injection, or (3) unperturbed. The sequencing was outsourced and the data received in 2013-14. Identification and validation of targets and pathways affected by both up- and down-regulation are ongoing. A genetic armc5 mutant generated by the Sanger TILLING project was obtained and has been bred to homozygosity. The above studies will include validation in these mutants. RNA expression constructs were obtained and antisense probes generated for in situ RNA stains of genes whose expression marks the zebrafish interrenal primordium, a tissue that is the functional equivalent to the human adrenal gland. In situ staining of zebrafish armc5 RNA was also performed and protocol optimization was initiated for co-localization studies to determine whether armc5 is expressed in the inter-renal primordium. This project will continue into 2015-16. Modeling copper deficiency-associated distal motoneuropathy The Menkes' gene on Xq13.3 encodes ATP7A, a P&#8208;type cation transporting ATPase localized to the plasma membrane and the trans&#8208;Golgi network (TGN) and critical for proper intracellular copper distribution. Two ATP7A missense mutations, T994I and P1386S cause a milder syndrome than Menkes that is still debilitating to children and young adults. In 2013-14, a project was initiated to clarify the structure-function relationship of ATP7A and motor neuron defects. A zebrafish line carrying mutations in the orthologous atp7a gene and a transgenic line that expresses GFP in motor neurons were obtained from outside sources and crossed together in anticipation of experiments that will look for motor neuron defects in atp7a mutants, which were documented in 2014-15. In 2014-15, RNA expression constructs were also built in anticipation of structure-function assays that will compare the ability of synthetic RNA from WT and mutant alleles to rescue motor neuron defects as well as other defects in atp7a null embryos. This project was awarded a Bench-to-Bedside grant, with $10,000 per annum funding to the NICHD Zebrafish Core through FY 2016-17 and the project will continue into 2015-16. Assessing human metastatic cell behaviors in a whole-body (zebrafish embryo) microenvironment. The dual goal of this project is (1) to determine the trophic range of certain metastatic melanoma and breast carcinoma cell lines and (2) to document cellular dynamics during early tumor formation from metastatic cells that have seeded into new microenvironments. In 2013-14, we optimized delivery of cells into the anterior CNS of embryos (for tumor formation studies) and the delivery of cells into the circulatory system (for trophism studies). During 2014-15, a substantial quantity of trophism data was collected. We also imported optically transparent and immunocompromised zebrafish lines that will enable the observation of tumor behaviors for longer periods of time. This project will continue in 2015-16. Finding neuroprotective drugs to mitigate hyperammonemia, a consequence of urea cycle defects & liver failure. Exposure of the brain to high ammonia causes neuro&#8208;cognitive deficits, intellectual disabilities, coma and death. In 2012-13 a strategy was developed in the core to use zebrafish embryos for identifying small molecules able to diminish the effects of hyperammonemia. The protocol was substantially improved in 2013-14 with the addition of an automated system for quantifying the spontaneous movement of experimental embryos. A library of hundreds of small molecules with known safety profiles for humans was screened in 2013-2015 and several promising candidates were identified for follow-up validation studies in zebrafish and other animal models. This project will continue into 2015-16. Other projects In 2014-15, the NICHD Zebrafish Core assisted the Lippincott-Schwartz laboratory (NICHD) with the acquisition of transgenic fish lines that label different cells of the immune system, in preparation for their own project that will examine the intersection of regeneration and immunity. NICHD Zebrafish Core resources were also used by the Waterman laboratory (NHLBI) to examine how altering the level of selected cytoskeletal proteins affects cellular behaviors.